Examples of a method for measuring a concentration of a specific substance contained in a liquid sample include a method in which a current with oxidation or reduction of the specific substance is detected by using an electrochemical reaction, in particular a measuring method using an amperometric chemical sensor. In such a case, in actual, used is a method in which the concentration of the certain specific substance that is the measurement object is indirectly measured in such a manner that the action of an enzyme on a specific substance that is a measurement object is allowed to quantitatively produce an enzymatic reaction product thereof by the enzymatic reaction and the current associated with oxidation or reduction of the enzymatic reaction product is detected using the electrochemical reaction thereto. Concretely, an enzyme electrode including an enzyme film layer disposed on an electrode for use in the enzymatic reaction, for example, an immobilized enzyme electrode, in which an enzyme is immobilized on the electrode of platinum or carbon to form an immobilized enzyme film layer thereof, is used as the working electrode therein. A predetermined bias is applied between the working electrode and a reference electrode, and the electrochemical reaction to said product, which has been obtained from the specific substance contained in the liquid sample by the function of the enzymatic reaction, is initiated by the bias applied between the working electrode and the reference electrode so as to generate a current in quantitative relation with an amount of the enzymatic reaction product by using the electrochemical reaction.
The chemical sensor using the enzyme electrode is immersed in the liquid sample, usually, an aqueous solution sample for use. When the aqueous solution sample permeates and/or penetrates into enzyme film layer, a phenomenon in which foreign matters and impurities contaminating the sample are adsorbed on the surface of the enzyme film layer or a phenomenon in which the surface of the electrode underlying the enzyme film layer for the enzyme electrode is polluted or degenerated sometimes occurs. When the foreign matters or impurities are adsorbed on the surface of the enzyme film layer, it is a factor for lowering efficiency of the enzymatic reaction with the specific substance that is the measurement object. This is also a factor for gradually deteriorating a ratio of the current amount (sensor sensitivity) measured in relation to the specific substance concentration with an elapse of time. On the other hand, even when the efficiency of the enzymatic reaction is maintained, once the electrode surface has been contaminated and changed in properties, the efficiency of the electrochemical reaction for measuring the enzymatic reaction product is influenced thereby. As a result, it is another factor for deteriorating the ratio of the current amount (sensor sensitivity) measured in relation to the specific substance concentration with the elapse of time.
Various methods have been proposed as a method of recovering the sensor sensitivity drop occurring in course of usage of the amperometric chemical sensor using the above-described enzyme electrode, for example, induced by the contamination and degeneration of the surface of the electrode for use in the working electrode. One of the methods proposed is a method for the case that the amperometric chemical sensor using the enzyme electrode is used, wherein at every stage post to usage for some predetermined period, a bias in a direction reverse to that of the bias usually applied between the working electrode and a counter electrode at the time of measurement is applied between the working electrode and the counter electrode for a short time, and accordingly, the contamination and degeneration of the electrode surface are removed to reactivate the enzyme electrode; as being proposed in Japanese Patent Application Laid-Open Nos. 57-060255, 60-155959, and 1-15649.
Additionally, in such a method in which at every stage post to usage for some predetermined period, the bias in the reverse direction between the working electrode and the counter electrode is applied for the short time, in some case depending on a chosen level for the reverse bias applied, it leads to such condition that electrochemical generation of hydrogen gas is resulted in an aqueous buffer solution in which the chemical sensor is stored, and the fine hydrogen bubbles generated adhere on the surface of the electrode used as the working electrode for the enzyme electrode. Alternatively, an overcurrent sometimes flows through the electrodes. In such a case, the surface of the electrode for use in the working electrode is occasionally damaged by the overcurrent.
A method for improving various defects of the method in which the reverse bias is applied for the short time is also proposed in Japanese Patent Publication No. 4-54175 as the method for recovering the aforementioned sensitivity drop in the chemical sensor that is induced in association with the repeated measurements. In the method disclosed in the publication, for the amperometric chemical sensor using the enzyme electrode, triangular-wave bias sweeping is performed with respect to the bias applied between the working electrode (enzyme electrode) and the counter electrode after the measurement to reactivate the enzyme electrode. Accordingly, the improvement against the drop of the sensor sensitivity with the elapse of time is achieved.
For example, as shown in FIG. 8, as for a measurement system using a chemical sensor being composed of, in a cell 101 having an inflow port and outflow port, a working electrode 103 using an enzyme electrode comprising a Pt electrode on the surface of which an enzyme film 102 is immobilized, and a counter electrode 104 consisting of the Pt electrode, in a condition in which the cell 101 is filled with the buffer solution that does not contain a substrate for the enzymatic reaction, the bias is applied between the working electrode 103 and counter electrode 104, at the time of measurement, in such a manner that the counter electrode 104 is grounded, and a bias of +0.6 V set on the basis of a saturated calomel electrode (SCE) used as reference is applied for the working electrode 103. When a sample liquid is flowed through the cell 101 at a constant flow rate, an enzymatic reaction product is produced from a specific substance (enzyme substrate material) present in the sample liquid with the enzymatic reaction in the enzyme film 102, the enzymatic reaction product causes the electrochemical reaction, and a response current thereof flows in said applied bias. Since a difference between the response current and a basal current observed at the time of the flow of the buffer solution is proportional to an amount of the enzymatic reaction product, and thus is also proportional to the amount of the specific substance (enzyme substrate material) involved in the enzymatic reaction, the concentration of the specific substance (enzyme substrate material) present in the sample liquid is quantified based on a calibration curve prepared beforehand. After the measurement, the buffer solution is flushed in the cell 101 to wash up the cell. As a result, the enzyme electrode system returns to an initial state. When this operation for cleaning up is repeated, measurements for different sample liquids are repeatedly carried out.
When such repetition of measurement is progressed, components, having a comparatively high molecular weight, such as protein or lipid, which are other than the specific substance (enzyme substrate material) that is the measurement object, adhere slightly to the surface of the enzyme film layer. Moreover, components having a comparatively low molecular weight, such as low molecular weight amine or organic acid penetrate or permeate into the inside of the enzyme film layer, and are adsorbed on the electrode surface, or an oxide coat film is sometimes formed on the electrode surface. In the method proposed in the Japanese Patent Publication No. 4-54175, for example., when a platinum electrode is used both for the working electrode and counter electrode, the applied bias is swept repeatedly through such an applied bias range that electrolysis of water molecules or oxidation/reduction reaction of the components or a support electrolyte in the buffer solution does not occur in the buffer solution for use, for example, in a range of −0.5 V to +1.3 V (applied bias set on the basis of SCE), in such a manner that the applied bias is first increased to an upper limit bias from an applied bias of +0.6 V at the time of the measurement at a sweeping rate of 0.1 to 1 V/s, and then the applied bias is decreased to a lower limit bias, and thereafter the triangular wave bias sweeping is continued between the lower and upper limit bias for a certain duration. Finally, after repeating the triangular-wave bias sweeping, the triangular wave bias sweeping is ended at a time when the applied bias reaches +0.6 V that is the initial applied bias used for measurement. When a reactivating treatment of the enzyme electrode by the triangular wave bias sweeping is performed at every stage post to predetermined times of measurement, the sensor sensitivity that has dropped with the elapse of time is recovered. A state in which there is not any excessive drop of the sensor sensitivity can be maintained over a long period.
As shown in FIG. 9, when the triangular wave bias sweeping is performed between the upper and lower limit biases, and states in which forward/reverse bias is applied are alternately repeated, the components electrostatically adsorbed on the electrode surface at the time of the measurement are removed by switching of the bias. In addition, the oxide coat film formed on the platinum surface for use in the working electrode 103 is removed stepwisely in course of repeating the states in which forward/reverse bias is applied alternatively.